肝靶向肽-人内皮抑制素融合蛋白表达条件的优化及活性鉴定

Optimization of expression conditions and activity identification of hepatocyte-targeting peptide-human endostatin

摘要:

为了获得足够多纯度高且有活性的肝靶向肽-人内皮抑制素融合蛋白（HTP-rES），首先研究了BL21/pET21b-HTP-rES重组菌株的生长曲线和最佳诱导时机；单因素分析不同pH值、不同诱导时间、不同诱导剂浓度、不同诱导温度时融合蛋白表达量；通过包涵体洗涤、复性、纯化，以获得高纯度的肝靶向肽-人内皮抑制素融合蛋白；最后采用流式细胞仪和MTT对融合蛋白进行活性鉴定。结果表明，BL21/pET21b-HTP-rES重组菌株在1.5–3.5 h处于对数生长期，培养基pH 8.0、IPTG终浓度0.06 mmol/L、42 ℃、诱导表达5 h为最佳表达条件。包涵体洗涤后纯度达60%，经复性、纯化后获得的目的蛋白纯度达到95%以上，对人肝癌细胞具有靶向性，能抑制人脐静脉内皮细胞的增殖。研究确立了融合蛋白最佳表达条件以及复性、纯化条件，为进一步研究其生物学活性及药物开发奠定基础。

Abstract:

To obtain sufficient purified and active fusion protein-hepatocyte-targeting peptide-human endostatin (HTP-rES), we studied the growth curve and the optimal induction timing of BL21/pET21b-HTP-rES. Different conditions of pH value, induction time, induction concentration and induction temperature were optimized by univariate analysis. After washing, refolding and purifying, the activity of fusion protein was identified by flow cytometry and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT). Results show that the logarithmic growth phase of BL21/pET21b-HTP-rES was from 1.5 h to 3.5 h, the optimum expression conditions were pH 8.0, 0.06 mmol/L IPTG, at 42 ℃ for 5 h. The purity of inclusion bodies was up to 60% after washing. The purity of target protein was more than 95% after refolding and purification. Our findings provide the foundation for further biological activity and drug development.